Myocardial infarction risk is increased by periodontal pathobionts: a cross-sectional study

To establish the role of periodontal pathobionts as a risk factor for myocardial infarction, we examined the contribution of five periodontal pathobionts and their virulence genes’ expressions to myocardial injury (Troponin-I) and coronary artery disease burden (SYNTAX-I scores) using hierarchical linear regression. Pathobiont loads in subgingival-plaques and intra-coronary-thrombi were compared. Troponin-I release increased with one 16S rRNA gene copy/ng DNA of Porphyromonas gingivalis (β = 6.8 × 10–6, 95% CI = 1.1 × 10–7–2.1 × 10–5), one-fold increased expressions of fimA (β = 14.3, 95% CI = 1.5–27.1), bioF-3 (β = 7.8, 95% CI = 1.1–12.3), prtH (β = 1107.8, 95% CI = 235.6–2451.3), prtP (β = 6772.8, 95% CI = 2418.7–11,126.9), ltxA (β = 1811.8, 95% CI = 217.1–3840.8), cdtB (β = 568.3, 95% CI = 113.4–1250.1), all p < 0.05. SYNTAX-I score increased with one 16S rRNA gene copy/ng DNA of Porphyromonas gingivalis (β = 3.8 × 10–9, 95% CI = 3.6 × 10–10-1.8 × 10–8), one-fold increased expressions of fimA (β = 1.2, 95% CI = 1.1–2.1), bioF-3 (β = 1.1, 95% CI = 1–5.2), prtP (β = 3, 95% CI = 1.3–4.6), ltxA (β = 1.5, 95% CI = 1.2–2.5), all p < 0.05. Within-subject Porphyromonas gingivalis and Tannerella forsythia from intra-coronary-thrombi and subgingival-plaques correlated (rho = 0.6, p < 0.05). Higher pathobiont load and/or upregulated virulence are risk factors for myocardial infarction. Trial registration: ClinicalTrials.gov Identifier: NCT04719026.


Section 1: Quantitative polymerase chain reaction (qPCR) for detection and quantification of periodontal bacterial load
Quantitative polymerase chain reaction assays were conducted on genomic bacterial DNA purified from all clinical samples. All samples were analysed in triplicate using 384 well plates (Sigma-Aldrich, Dorset, UK). Each well contained a 10 µL reaction mixture that included: 5 µL of 2 × Takyon qPCR dTTP MasterMix (Eurogentec, Hampshire, UK), 1 µL of template genomic DNA, 0.5 µL of forward and reverse primers each, 0.2 µL of the probe (Eurogentec, Hampshire, UK) and 2.8 µL nuclease-free water. The primer and probe sequences are listed in Supplementary Table 1. The primer (500 nM) and probe (100 nM) concentrations were optimised in a pilot assay (data not shown). In each plate, genomic DNA of P. gingivalis 33277 was used as a positive control and nuclease-free water (Sigma-Aldrich, Dorset, UK) as a negative control. All the qPCR assays were performed on a LightCycler 480 Real-Time PCR (Roche Molecular Systems, West Sussex, UK) device. The thermocycling program was set for: 10min at 95 °C (initial denaturation), 40 cycles of 30 s at 95 °C, 1 min at 60 °C. The increase in fluorescence was monitored during PCR amplification and all data was analysed using LightCycler® 480 Software (Roche Molecular Systems, West Sussex, UK). The absolute quantification method 28 was utilised to quantify the bacterial load of all selected periodontal bacterial species. P. gingivalis (ATCC 33277), a previously characterised T. forsythia isolate, A. actinomycetemcomitans isolated from a periodontitis patient at Aberdeen Dental Hospital and P. intermedia (ATCC 25611) were used as reference strains to construct the respective standard curves (Supplementary Figure 3).

Section 2: Reverse transcription PCR (RT-qPCR) for quantification of the expression of virulence genes
RT-PCR assays were conducted on purified RNA extracted from subgingival plaque and tested in triplicate. The expression levels of three genes per PCR-well were multiplexed using a combination of three dual labelled probes as follows: i) channel 1: 6 FAM and Eclipse Dark Quencher, ii) channel-2: HEX and Eclipse Dark Quencher, iii) channel-3: Cy5 and DDQII. Each PCR-well contained: 5 µL of 2× Takyon qPCR dTTP MasterMix (Eurogentec, Hampshire, UK), 0.25 µL of forward and reverse primers of 3 distinct target genes, respectively; 0.1 µL of probes of 3 distinct target genes, respectively; 1 µL of the template (RNA), 0.1 µL of Takyon One-Step Kit Converter (Eurogentec, Hampshire, UK) and 2.1 µL of nuclease-free water. The primer and probe sequences targeting virulence genes are listed in Supplementary Table 3. The concentrations of primers (500 nM) and probes (100 nM) were optimised in a pilot assay (data not shown). Each plate contained purified P. gingivalis 33277 RNA as a positive control and nuclease-free water (Sigma-Aldrich, Dorset, UK) as a negative control. All RT-qPCR assays were performed in a LightCycler 480 Real-Time PCR (Roche Molecular Systems, West Sussex, UK). The thermocycling program was: i) step-1 (reverse transcription):10 min at 48 °C, ii) step-2 (cDNA amplification): 10 min at 95 °C (initial denaturation), 40 cycles of 30 s at 95 °C, 1 min at 60 °C. The increase in fluorescence was monitored during PCR amplification and all data was analysed using LightCycler 480 Software (Roche Molecular Systems, West Sussex, UK). The expression levels were normalised against 3 reference genes (recA, glyA and groL) for relative quantification using the CT method 30 .

Section 3: Enzyme-linked immunosorbent assay (ELISA) to quantify different anti-P. gingivalis antibody isotypes
High binding polystyrene 96-well plates were coated with P. gingivalis-purified lipopolysaccharide (InvivoGen, Toulouse, France) at 1µg/mL concentration and stored overnight at 4 °C. Thereafter, plates were blocked with 5 % bovine serum albumin in PBS at room temperature for 30 minutes. Each serum and saliva sample at 1:10 dilution was incubated in duplicate for 2 hours at room temperature. The bound serum IgG, IgM and IgA antibodies were detected by horseradish peroxidase-conjugated, anti-human IgG (A0170, Sigma-Aldrich, Dorset, UK) diluted at 1:30000, antihuman IgM (A6907, Sigma-Aldrich, Dorset, UK) diluted at 1:10000, and anti-human IgA (A18781, Thermo Fisher Scientific) at 1:2500 concentrations, respectively. Similarly, for the plate containing saliva samples, an anti-human IgA2 antibody (BT91-4005, Generon, Dublin, Ireland) was added at 1:1000 concentration. Samples were visualised spectrophotometrically at 492 nm using o-Phenylenediamine dihydrochloride (SIGMAFAST OPD, P9187 Sigma-Aldrich, Dorset, UK). The dilution factors for test sera, saliva samples and HRP-conjugated antibodies were optimised before the main experiment (data not shown). Spectrophotometry values were corrected for non-specific binding of the HRP-conjugated.  • has a potent proteolytic activity and accounts for 85% P. gingivalis-induced tissue breakdown 15 ;
fimA Major fimbrium subunit FimA type-2 • enhances bacterial adhesion to multiple types of surfaces, such as the extracellular matrix, host cells and other bacteria-critical for biofilm formation 15 ; • fimA genotypes II and IV are frequently detected in patients with periodontitis and have strong epithelial cell adhesion and invasion properties 15 and fimA type II genotype is most prevalent in coronary arterial atheromatous plaque samples from patients with periodontitis 15 ; • activates gingival fibroblasts, monocyte and macrophages via TLR2 pathway to induce the production of TNF-α, IL-1α, IL-1β, and IL-6 cytokines 15 ;
prtH PrtH protease • causes detachment of epithelial cells and cytopathic activity arrest cells in their G2 phase 17 and increases the mitochondrial oxidative membrane potential in cells, leading to IL-8 production from detached fibroblast cells and neutrophil recruitment 17 ; • prtH genotype levels are strongly associated with advanced periodontal attachment loss compared to periodontally-healthy subjects 17 .

bspA
Surface antigen BspA • involves in protein-protein interactions, signal transduction, bacterial adherence and invasion into epithelial cells 17 ; • triggers TLR-2-dependent release of bone-resorbing pro-inflammatory cytokines from monocytes and chemokine IL-8 from gingival epithelial cells 17 ; • binds to the extracellular matrix component fibronectin and the clotting factor fibrinogen 17 ; • T. forsythia mutant defective in the expression of BspA protein showed significantly less bone loss as compared to mice infected with the wild-type strain 17 .

msp
Major outer sheath protein • has a potent pore-forming cytotoxic activity towards periodontal ligament epithelial cells, causing cell detachment and apoptosis 16 ; • blocks the IL-8 production required for neutrophil chemotaxis and phagocytic and thereby evade the host immune response 16 ; • degrades host cell protease inhibitors and fibronectin 16 ; • an immunodominant antigen of T. denticola that elicits a strong antibody response 16 .

ltxA
Leukotoxin A • the large pore-forming toxin that binds to the lymphocyte function-associated receptor 1 (LFA-1) and disrupts the membrane integrity of most of the immune cells, leading to cell death and immune evasion 18 ; • activates neutrophil degranulation causing a massive release of lysosomal enzymes, net-like structures, and matrix metalloproteinases (MMPs) and induces apoptosis in lymphocytes 18 ; • activates inflammasome complex in the monocytes/macrophages, including the cysteine proteinase caspase-1, which induces activation and secretion of the pro-inflammatory cytokines IL-1β and IL-18 18 ; • frequently detected with serotype b and JP2 isolates that are strongly associated with rapid periodontal attachment loss 18 . cdtB Cytolethal distending toxin subunit B • part of a tripartite complex comprised of subunits CdtA, CdtB, and CdtC, while CdtB protein is the active subunit. It has a potent type I DNase activity causing G2 phase cell arrest of a variety of proliferating cells, including epithelial cells, fibroblasts, human periodontal ligament cells, and lymphocytes, leading to cell apoptosis 18 ; • targets and invades the immune responses by inducing apoptosis of non-proliferative monocytic cells and T lymphocytes 18 ; • stimulates the production of pro-inflammatory cytokines by peripheral blood mononuclear cells, such as interferon (IFN)-γ, Interleukin (IL)-1β, IL-6, and IL-8; a virulence property potentially independent of the toxin's type I DNase activity 18 ; • stimulates RANKL production by periodontal fibroblasts and periodontal ligament cells as well as T-cells, leading to bone loss 18 .

clpB
Chaperone protein ClpB • ATP-dependent chaperone protein that is involved in the quorum sensing system of periodontal microbial biofilm and aiding coaggregation of P.
gingivalis by upregulating its virulence factors, such as iron uptake, flagellar protein synthesis and gingipain production 29 .
dnaK Chaperone protein DnaK • stress or heat shock protein (hsp70) is up-regulated during infection and involved in conserved cellular processes, such as protein-folding reactions and the assembly/disassembly of protein complexes 29 ; • anti-hsp70 antibodies cross-react with HSPs expressed on the endothelial cells and thereby contribute to the atherosclerosis process 29 . Supplementary Figure 1 The log10 transformed serially diluted bacterial loads and the corresponding quantification cycle (Cq) values generated in qPCR assay plotted for: A) P. gingivalis, B) T. forsythia, C) A. actinomycetemcomitans and D) P. intermedia. The linear regression equations generated by each standard curve were used to calculate the bacterial load of the respective species in the tested samples. R 2 = squared correlation coefficient. The red dotted red lines in each plot represent the upper cut-off limit of 35 Cq, set for bacterial detection.